-
Scientific Reports Nov 2022Immunotherapy has a number of advantages over traditional anti-tumor therapy but can cause severe adverse reactions due to an overactive immune system. In contrast, a...
Immunotherapy has a number of advantages over traditional anti-tumor therapy but can cause severe adverse reactions due to an overactive immune system. In contrast, a novel metabolic treatment approach can induce metabolic vulnerability through multiple cancer cell targets. Here, we show a therapeutic effect by inducing nucleotide imbalance and apoptosis in triple negative breast cancer cells (TNBC), by treating with cytosolic thymidylate 5'-phosphohydrolase (CT). We show that a sustained consumption of dTMP by CT could induce dNTP imbalance, leading to apoptosis as tricarboxylic acid cycle intermediates were depleted to mitigate this imbalance. These cytotoxic effects appeared to be different, depending on substrate specificity of the 5' nucleotide or metabolic dependency of the cancer cell lines. Using representative TNBC cell lines, we reveal how the TNBC cells were affected by CT-transfection through extracellular acidification rate (ECAR)/oxygen consumption rate (OCR) analysis and differential transcription/expression levels. We suggest a novel approach for treating refractory TNBC by an mRNA drug that can exploit metabolic dependencies to exacerbate cell metabolic vulnerability.
Topics: Humans; Triple Negative Breast Neoplasms; Thymidine Monophosphate; Cell Line, Tumor; Apoptosis; Phosphoric Monoester Hydrolases
PubMed: 36414668
DOI: 10.1038/s41598-022-24706-4 -
Analytica Chimica Acta Dec 2022Plasmonic micro/nanobeads exhibit unique physicochemical properties attributed to their localized surface plasmon resonance (LSPR), enabling use in sensitive suspension...
Plasmonic micro/nanobeads exhibit unique physicochemical properties attributed to their localized surface plasmon resonance (LSPR), enabling use in sensitive suspension array assays and matrix-assisted laser deposition/ionization mass spectrometry (MALDI-MS) analysis. Herein, we report a facile method for the preparation of magnetic plasmonic micro/nanobeads by the combination of Shirasu porous glass (SPG) membrane emulsification and polydopamine (PDA)-assisted in-situ reduction. The magnetic responsiveness properties endowed by doped FeO nanoparticles result in easy and complete separation of unwanted components during the preparation and bio-reaction processes. In addition, the coverage degree of the plasmonic shell can be flexibly controlled. As a result of the significant metal-enhanced fluorescence effect, as-prepared plasmonic microbeads enable the sensitive detection of alpha-fetoprotein (AFP) and deoxyribonucleotide (DNA) in suspension array with detection limits of 0.11 ng mL and 1.65 fmol mL, respectively, 8.6 times and 2 orders of magnitude higher than unmodified microbeads. Furthermore, as-prepared plasmonic nanobeads can be used as a matrix for MALDI-MS to allow the detection of low molecular weight biological molecules. As little as 0.2 pmol of proline and serine can be detected in a sample as small as 0.5 μL. This work provides a general strategy for the design of multifunctional plasmonic micro/nanomaterials that will help promote further advancements in sample analysis.
Topics: Surface Plasmon Resonance; Biological Assay; Nanoparticles; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Nanostructures
PubMed: 36396232
DOI: 10.1016/j.aca.2022.340577 -
International Journal of Molecular... Nov 2022The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering....
The genome editing approach using the components of the CRISPR/Cas system has found wide application in molecular biology, fundamental medicine and genetic engineering. A promising method is to increase the efficacy and specificity of CRISPR/Cas-based genome editing systems by modifying their components. Here, we designed and chemically synthesized guide RNAs (crRNA, tracrRNA and sgRNA) containing modified nucleotides (2'-O-methyl, 2'-fluoro, LNA-locked nucleic acid) or deoxyribonucleotides in certain positions. We compared their resistance to nuclease digestion and examined the DNA cleavage efficacy of the CRISPR/Cas9 system guided by these modified guide RNAs. The replacement of ribonucleotides with 2'-fluoro modified or LNA nucleotides increased the lifetime of the crRNAs, while other types of modification did not change their nuclease resistance. Modification of crRNA or tracrRNA preserved the efficacy of the CRISPR/Cas9 system. Otherwise, the CRISPR/Cas9 systems with modified sgRNA showed a remarkable loss of DNA cleavage efficacy. The kinetic constant of DNA cleavage was higher for the system with 2'-fluoro modified crRNA. The 2'-modification of crRNA also decreased the off-target effect upon in vitro dsDNA cleavage.
Topics: CRISPR-Cas Systems; Endonucleases; Gene Editing; Nucleotides; RNA, Small Untranslated
PubMed: 36362256
DOI: 10.3390/ijms232113460 -
ACS Chemical Biology Nov 2022Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and...
Primase-DNA polymerase (PrimPol) is involved in reinitiating DNA synthesis at stalled replication forks. PrimPol also possesses DNA translesion (TLS) activity and bypasses several endogenous nonbulky DNA lesions in vitro. Little is known about the TLS activity of PrimPol across bulky carcinogenic adducts. We analyzed the DNA polymerase activity of human PrimPol on DNA templates with seven -dG lesions of different steric bulkiness. In the presence of Mg ions, bulky -isobutyl-dG, -benzyl-dG, -methyl(1-naphthyl)-dG, -methyl(9-anthracenyl)-dG, -methyl(1-pyrenyl)-dG, and -methyl(1,3-dimethoxyanthraquinone)-dG adducts fully blocked PrimPol activity. At the same time, PrimPol incorporated complementary deoxycytidine monophosphate (dCMP) opposite -ethyl-dG with moderate efficiency but did not extend DNA beyond the lesion. We also demonstrated that mutation of the Arg288 residue abrogated dCMP incorporation opposite the lesion in the presence of Mn ions. When Mn replaced Mg, PrimPol carried out DNA synthesis on all DNA templates with -dG adducts in standing start reactions with low efficiency and accuracy, possibly utilizing a lesion "skipping" mechanism. The TLS activity of PrimPol opposite -ethyl-dG but not bulkier adducts was stimulated by accessory proteins, polymerase delta-interacting protein 2 (PolDIP2), and replication protein A (RPA). Molecular dynamics studies demonstrated the absence of stable interactions with deoxycytidine triphosphate (dCTP), large reactions, and C1'-C1' distances for the -isobutyl-dG and -benzyl-dG PrimPol complexes, suggesting that the size of the adduct is a limiting factor for efficient TLS across minor groove adducts by PrimPol.
Topics: Humans; Deoxycytidine Monophosphate; DNA Damage; Deoxyguanosine; DNA Replication; DNA-Directed DNA Polymerase; DNA; DNA Adducts; Nuclear Proteins; DNA Primase; Multifunctional Enzymes
PubMed: 36318733
DOI: 10.1021/acschembio.2c00717 -
Current Opinion in Structural Biology Dec 2022Ribonucleotide reductases (RNRs) use radical-based chemistry to convert ribonucleotides into deoxyribonucleotides, an essential step in DNA biosynthesis and repair.... (Review)
Review
Ribonucleotide reductases (RNRs) use radical-based chemistry to convert ribonucleotides into deoxyribonucleotides, an essential step in DNA biosynthesis and repair. There are multiple RNR classes, the best studied of which is the class Ia RNR that is found in Escherichia coli, eukaryotes including humans, and many pathogenic and nonpathogenic prokaryotes. This review covers recent advances in our understanding of class Ia RNRs, including a recent reporting of a structure of the active state of the E. coli enzyme and the impacts that the structure has had on spurring research into the mechanism of long-range radical transfer. Additionally, the review considers other recent structural and biochemical research on class Ia RNRs and the potential of that work for the development of anticancer and antibiotic therapeutics.
Topics: Humans; Ribonucleotide Reductases; Escherichia coli
PubMed: 36272229
DOI: 10.1016/j.sbi.2022.102489 -
Molecules (Basel, Switzerland) Sep 2022In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene...
In 2002, a new class of thymidylate synthase (TS) involved in the de novo synthesis of dTMP named Flavin-Dependent Thymidylate Synthase (FDTS) encoded by the thyX gene was discovered; FDTS is present only in 30% of prokaryote pathogens and not in human pathogens, which makes it an attractive target for the development of new antibacterial agents, especially against multi-resistant pathogens. We report herein the synthesis and structure-activity relationship of a novel series of hitherto unknown pyrido[1,2-e]purine-2,4(1H,3H)-dione analogues. Several synthetics efforts were done to optimize regioselective N1-alkylation through organopalladium cross-coupling. Modelling of potential hits were performed to generate a model of interaction into the active pocket of FDTS to understand and guide further synthetic modification. All those compounds were evaluated on an in-house in vitro NADPH oxidase assays screening as well as against Mycobacterium tuberculosis ThyX. The highest inhibition was obtained for compound 23a with 84.3% at 200 µM without significant cytotoxicity (CC50 > 100 μM) on PBM cells.
Topics: Anti-Bacterial Agents; Dinitrocresols; Flavins; Humans; Mycobacterium tuberculosis; NADPH Oxidases; Purines; Structure-Activity Relationship; Thymidine Monophosphate; Thymidylate Synthase
PubMed: 36234754
DOI: 10.3390/molecules27196216 -
Aging Oct 2022Ribonucleotide reductase (RNR) small subunit M2 (RRM2) levels are known to regulate the activity of RNR, a rate-limiting enzyme in the synthesis of deoxyribonucleotide...
Ribonucleotide reductase (RNR) small subunit M2 (RRM2) levels are known to regulate the activity of RNR, a rate-limiting enzyme in the synthesis of deoxyribonucleotide triphosphates (dNTPs) and essential for both DNA replication and repair. The high expression of RRM2 enhances the proliferation of cancer cells, thereby implicating its role as an anti-cancer agent. However, little research has been performed on its role in the prognosis of different types of cancers. This pan-cancer study aimed to evaluate the effect of high expression of RRM2 the tumor prognosis based on clinical information collected from The Cancer Genome Atlas (TCGA) and The Genotype-Tissue Expression (GTEx) databases. We found RRM2 gene was highly expressed in 30 types of cancers. And we performed a pan-cancer analysis of the genetic alteration status and methylation of RRM2. Results indicated that RRM2 existed hypermethylation, associated with m6A, m1A, and m5C related genes. Subsequently, we explored the microRNAs (miRNA), long non-coding RNAs (lncRNA), and the transcription factors responsible for the high expression of RRM2 in cancer cells. Results indicated that has-miR-125b-5p and has-miR-30a-5p regulated the expression of RRM2 along with transcription factors, such as CBFB, E2F1, and FOXM. Besides, we established the competing endogenous RNA (ceRNA) diagram of lncRNAs-miRNAs-circular RNAs (circRNA) involved in the regulation of RRM2 expression. Meanwhile, our study demonstrated that high-RRM2 levels correlated with patients' worse prognosis survival and immunotherapy effects through the consensus clustering and risk scores analysis. Finally, we found RRM2 regulated the resistance of immune checkpoint inhibitors through the PI3K-AKT single pathways. Collectively, our findings elucidated that high expression of RRM2 correlates with prognosis and tumor immunotherapy in pan-cancer. Moreover, these findings may provide insights for further investigation of the RRM2 gene as a biomarker in predicting immunotherapy's response and therapeutic target.
Topics: Humans; Ribonucleoside Diphosphate Reductase; RNA, Long Noncoding; RNA, Circular; Computational Biology; Immune Checkpoint Inhibitors; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Prognosis; MicroRNAs; Neoplasms; Immunotherapy; Transcription Factors; Deoxyribonucleotides; Gene Expression Regulation, Neoplastic; Cell Line, Tumor
PubMed: 36202136
DOI: 10.18632/aging.204315 -
Aging Sep 2022The expression of deoxythymidylate kinase (DTYMK) is up-regulated in liver cancer. However, the underlying biological function and potential mechanisms of DTYMK driving...
The expression of deoxythymidylate kinase (DTYMK) is up-regulated in liver cancer. However, the underlying biological function and potential mechanisms of DTYMK driving the progression of lung adenocarcinoma remains unclear. In this study, we investigated the role of DTYMK in lung adenocarcinoma and found that the expression of DTYMK in LUAD tissues was significantly higher than that of DTYMK expression in adjacent normal tissues. Kaplan-Meier survival analysis showed that patients with higher DTYMK expression correlated with adverse prognosis. ROC curve analysis showed that the AUC value of DTYMK was 0.914. Correlation analysis showed that DTYMK expression was associated with immune infiltration in LUAD. Finally, we determine that DTYMK regulated cell proliferation, cell migration, and cell cycle of lung adenocarcinoma . In conclusion, our data demonstrated that DTYMK was correlated with progression and immune infiltration, and could serve as a prognostic biomarker for lung adenocarcinoma.
Topics: Humans; Thymidine Monophosphate; Lung Neoplasms; Adenocarcinoma of Lung; Prognosis; Biomarkers; Tumor Microenvironment
PubMed: 36170019
DOI: 10.18632/aging.204308 -
Sub-cellular Biochemistry 2022Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to... (Review)
Review
Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to deoxyribonucleotides. This chapter primarily presents an overview of this multifaceted and complex enzyme, covering RNR's role in enzymology, biochemistry, medicinal chemistry, and cell biology. It further focuses on RNR from mammals, whose interesting and often conflicting roles in health and disease are coming more into focus. We present pitfalls that we think have not always been dealt with by researchers in each area and further seek to unite some of the field-specific observations surrounding this enzyme. Our work is thus not intended to cover any one topic in extreme detail, but rather give what we consider to be the necessary broad grounding to understand this critical enzyme holistically. Although this is an approach we have advocated in many different areas of scientific research, there is arguably no other single enzyme that embodies the need for such broad study than RNR. Thus, we submit that RNR itself is a paradigm of interdisciplinary research that is of interest from the perspective of the generalist and the specialist alike. We hope that the discussions herein will thus be helpful to not only those wanting to tackle RNR-specific problems, but also those working on similar interdisciplinary projects centering around other enzymes.
Topics: Animals; Deoxyribonucleotides; Mammals; Oxidoreductases; Ribonucleotide Reductases; Ribonucleotides
PubMed: 36151376
DOI: 10.1007/978-3-031-00793-4_5 -
Nutrients Sep 2022Methionine restriction and selenium supplementation are recommended because of their health benefits. As a major nutrient form in selenium supplementation,... (Comparative Study)
Comparative Study
Methionine restriction and selenium supplementation are recommended because of their health benefits. As a major nutrient form in selenium supplementation, selenomethionine shares a similar biological process to its analog methionine. However, the outcome of selenomethionine supplementation under different methionine statuses and the interplay between these two nutrients remain unclear. Therefore, this study explored the metabolic effects and selenium utilization in HepG2 cells supplemented with selenomethionine under deprived, adequate, and abundant methionine supply conditions by using nuclear magnetic resonance-based metabolomic and molecular biological approaches. Results revealed that selenomethionine promoted the proliferation of HepG2 cells, the transcription of selenoproteins, and the production of most amino acids while decreasing the levels of creatine, aspartate, and nucleoside diphosphate sugar regardless of methionine supply. Selenomethionine substantially disturbed the tricarboxylic acid cycle and choline metabolism in cells under a methionine shortage. With increasing methionine supply, the metabolic disturbance was alleviated, except for changes in lactate, glycine, citrate, and hypoxanthine. The markable selenium accumulation and choline decrease in the cells under methionine shortage imply the potential risk of selenomethionine supplementation. This work revealed the biological effects of selenomethionine under different methionine supply conditions. This study may serve as a guide for controlling methionine and selenomethionine levels in dietary intake.
Topics: Amino Acids; Aspartic Acid; Choline; Citrates; Creatine; Dietary Supplements; Glycine; Hep G2 Cells; Humans; Hypoxanthines; Lactates; Methionine; Nucleoside Diphosphate Sugars; Racemethionine; Selenium; Selenomethionine; Selenoproteins
PubMed: 36145081
DOI: 10.3390/nu14183705